Pulmonary microvascular endothelial cells (PMVECs) possess strongly adherent cell-cell junctions that are necessary to limit fluid, solute and macromolecule permeability into interstitial and alveolar compartments, which is important for efficient gas exchange. PMVEC junction strength is dynamically adjusted by intracellular cAMP concentrations. The type 6 adenylyl cyclase (AC6) synthesizes cAMP at the cell membrane. cAMP signaling is targeted to physiologically relevant effector molecules by type 4 phosphodiesterases (PDE4), specifically the -D4 isoform, which is membrane-localized by spectrin. The membrane-localized cAMP pool strengthens PMVEC barrier function. In contrast, Pseudomonas aeruginosa introduces a soluble adenylyl cyclase toxin, ExoY, into PMVECs that generates a cytosolic cAMP pool. cAMP synthesis within the cytosol disrupts, rather than strengthens, the PMVEC barrier. To determine whether membrane or cytosolic AC activity dominates in control of endothelial cell barrier function, we utilized a chimeric mammalian soluble AC enzyme that could be activated by forskolin. Simultaneous stimulation of membrane and cytosolic AC activity by forskolin disrupts, rather than strengthens, the PMVEC barrier, indicating soluble AC activity dominantly controls barrier strength. Preliminary data suggest soluble ACs associate with the centrosome and its associated microtubules, and may therefore reorganize microtubule architecture necessary to induce PMVEC gaps. Thus, this proposal tests the overall hypothesis that membrane-localized ACs produce a cAMP pool that strengthens, whereas cytosolic ACs produce a cAMP pool that disrupts, the PMVEC barrier. Specific Aims test the related Hypotheses that: [1] AC6 generates a membrane cAMP pool that is maintained by a spectrin and PDE4(D4) interaction;[2] Soluble ACs generate a cytosolic cAMP pool that controls microtubule organization;and, [3] cAMP that accesses the cytosolic compartment disassembles microtubules and disrupts the endothelial cell barrier. Completion of this work will contribute to our understanding of how cAMP acts to control PMVEC barrier strength, and will seek to resolve pathogenic mechanisms of bacteria like Pseudomonas aeruginosa, which utilize adenylyl cyclase toxins to disrupt the endothelial cell barrier and increase permeability.